Cryptosporidiosis caused by two distinct species in Russian tortoises anda pancake tortoise

Chris Griffin a, Drury R. Reavill b, Brian A. Stacy c, April L. Childress c, James F.X. Wellehan Jr.c,*a Griffin Avian and Exotic Veterinary Hospital, 2100 Lane Street, Kannapolis, NC 28083, USAb Zoo/Exotic Pathology Service, 7647 Wachtel Way, Citrus Heights, CA 95610, USAc College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA

Veterinary Parasitology 170 (2010) 14–19

A R T I C L E I N F O

Article history:

Received 12 October 2009

Received in revised form 21 January 2010

Accepted 26 January 2010

Keywords:

Agrionemys horsfieldii

Cryptosporidium

Malacochersus tornieri

Russian tortoise

Pancake tortoise

A B S T R A C T

Cryptosporidiosis in squamates is well documented, but there is very limited information

available on cryptosporidiosis in testudines. We describe three cases of cryptosporidiosis

in tortoises with associated pathology. Two Russian tortoises (Agrionemys [Testudo]

horsfieldii) and a pancake tortoise (Malacochersus tornieri), all from separate collections,

were found dead. At necropsy, two had histological evidence of intestinal cryptospor-

idiosis and one had gastric cryptosporidiosis. Consensus Cryptosporidium sp. PCR and

sequencing was used to identify the Cryptosporidium sp. present in these three tortoises. In

the juvenile Russian tortoise with gastric cryptosporidiosis, the organism had 98%

homology with a previously reported sequence from an Indian star tortoise isolate. A

second chelonian Cryptosporidium sp. was identified in the pancake tortoise and the

second Russian tortoise. This sequence was 100% identical to a shorter gene sequence

previously reported in a marginated tortoise. This is the first report coordinating pathology

with Cryptosporidium characterization in chelonians. The two Cryptosporidium sp. found in

tortoises segregate according to site of infection, and there may be further differences in

pathology, host range, and transmission. These Cryptosporidium sp. appear to be able to

infect diverse tortoise host species. This may be an under-recognized problem in tortoises.

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1. Introduction

Cryptosporidium sp. are well documented as commonand clinically significant pathogens of reptiles (Brownsteinet al., 1977; Cranfield and Graczyk, 1994; Terrell et al.,2003). The first documented and most well known speciescausing disease in non-avian reptiles is Cryptosporidium

serpentis. Experimental infections have shown that thisagent causes gastric hypertrophy with focal necrosis andpetechiation in snakes (Cranfield and Graczyk, 1994).Clinically, regurgitation and mid-body swelling are typicalantemortem signs.

* Corresponding author. Tel.: +1 352 392 2226; fax: +1 352 392 6436.

E-mail address: wellehanj@vetmed.ufl.edu (James F.X. Wellehan Jr.).

0304-4017/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2010.01.039

Cryptosporidium varanii (saurophilum) is the next bestdocumented species in this genus seen in non-avianreptiles. Commonly known to practitioners as C. saur-

ophilum, it has recently been shown that this name isactually a junior synonym for C. varanii (Pavlasek and Ryan,2008). It is found in the intestine and cloaca. Intestinalcryptosporidiosis in reptiles often presents as weight loss,anorexia, lethargy, and diarrhea (Terrell et al., 2003). Co-infections with other enteric pathogens such as adeno-viruses may exacerbate disease (Wellehan et al., 2004).

With the advent of molecular phylogeny, our under-standing of Cryptosporidium diversity has increasedgreatly. Traditional methods of Cryptosporidium speciationinvolved looking at host range and morphology. Moleculardata has corrected a number of errors that were made incoccidian phylogeny based on morphologic data. Thegenus Isospora was divided into Isospora and Cystoisospora,

C. Griffin et al. / Veterinary Parasitology 170 (2010) 14–19 15

showing that the presence or absence of Steida bodies,and avian or mammalian host specificity, is morephylogenetically informative than number of sporocystsor sporozoites for these genera (Barta et al., 2005). Thegenus Cryptosporidium is particularly challenging, as theylack truly distinctive morphologic criteria, making mor-phologic identification unreliable (Fall et al., 2003).

Beyond the named species using non-avian reptilehosts, additional distinct clades of Cryptosporidium havebeen identified using sequence-based methodologies. ACryptosporidium species was identified in a fecal samplefrom a viper boa (Candoia asper) and found to be distinctfrom other known reptile Cryptosporidium (Xiao et al.,2004). An identical organism was later found in 57 of 223wild Japanese grass snakes (Rhabdophis tigris), and wasassociated with mucosal edema, goblet cell loss, andscattered necrosis in the small intestine (Kuroki et al.,2008). Another Cryptosporidium has been found in the fecesof a Boa constrictor that is probably distinct at a specieslevel (Xiao et al., 2004). C. muris has also been seen inreptiles, but it is unclear whether this is infecting theanimals or passing through from ingested prey (Xiao et al.,2004). Cryptosporidium has also been associated with auralpolyps in green iguanas (Iguana iguana), but no speciesidentification has been done (Fitzgerald et al., 1998).

In hosts in the order Testudines (turtles and tortoises),data on Cryptosporidium is much more limited. In anEgyptian tortoise (Testudo kleinmanni), small intestinalCryptosporidium was associated with mixed inflammatoryinfiltrates, although molecular characterization of theorganism was not done (Graczyk et al., 1998b). ACryptosporidium has been found in the feces of Indian startortoises (Geochelone elegans) and Hermann’s tortoises(Testudo hermanii) that is probably distinct at a specieslevel, but no investigation on site of infection or associatedpathology has been done (Xiao et al., 2004; Alves et al.,2005; Pedraza-Dıaz et al., 2009). This organism has alsobeen reported from feces of a ball python (Python regius)(Pedraza-Dıaz et al., 2009).

Recently, another survey in Italy found a second distinctCryptosporidium in feces of a marginated tortoise (Testudo

marginata) that is probably distinct at a species level(Traversa et al., 2008). A near-identical sequence has beenfound in feces of a ball python and feces of a veiledchameleon (Chamaeleo calyptratus) (Pedraza-Dıaz et al.,2009). No investigation on site of infection or associatedhistopathology was done. Feces from several additionalTestudo sp. tortoises were found to contain the bovinegenotype of C. parvum (Traversa et al., 2008).

While a common and significant problem in squamates,cryptosporidiosis has not been frequently recognized intestudines. It is not known whether this reflects a lowerinfection or disease rate in testudines, or whether this isdue to lack of recognition. There is little data available, butone study found that 7 of 21 (33%) of tortoises in Italy hadCryptosporidium present in their feces (Traversa et al.,2008).

Specific identification of Cryptosporidium in coordinationwith pathologic investigation is crucial for understandingsignificance and epidemiology of infection. There has notbeen previous data on pathology associated with specific

Cryptosporidium in Testudines. This report describes pathol-ogy associated with two unnamed Cryptosporidium speciesin three tortoises.

2. Materials and methods

2.1. Clinical presentation and pathology

2.1.1. Case 1

A privately owned juvenile male Russian tortoise(Agrionemys [Testudo] horsfieldii) with excellent diet andhusbandry did not grow normally over an 18–20-monthperiod. Over that time, the tortoise only gained from 25 to34 g at his heaviest. The tortoise was afforded dailyexposure to sun during the warmer months in NorthCarolina (March–October) and fed a diet appropriate forthis species. Despite multiple negative fecal parasite examsby direct and flotation methods, and multiple adminis-trations of fenbendazole (25 mg/kg, q 7 days for 3treatments) and metronidazole (20 mg/kg PO q 24 h for7–10 days) as general anti-parasitic agents, the tortoisenever fully began to thrive and grow for the first 18months.

After a short period of lethargy and depression, thetortoise passed away and was necropsied. All tissuesamples obtained at necropsy were preserved in 10%neutral buffered formalin solution prior to being processedroutinely. Paraffin-embedded tissues were sectioned atapproximately 5 mm, mounted on glass slides, and stainedwith hematoxylin and eosin (HE). Representative sectionsof kidney, urinary bladder, adrenal gland, lung, trachea,brain, heart, ovary, stomach, intestines, and liver wereexamined.

2.1.2. Case 2

A female Russian tortoise from a different collection,presented for inappetence with pale mucous membranesand lethargy. It was euthanized and submitted fornecropsy. Tissues were collected and processed forhistologic examination similarly to case 1.

2.1.3. Case 3

A male pancake tortoise (Malacochersus tornieri), wasamong a large group of tortoises confiscated fromsmugglers (6 months prior to death) and was subsequentlytransferred with a female conspecific to a zoologicalinstitution. Both tortoises had been at the most recentfacility for 8 days. The male drank, but would not eat andbecame progressively weaker and lethargic over the 24 hpreceding death. Severe edema of the head and neck, andweakness of the forelimbs were noted near the time ofdeath. Tissues were collected and processed for histologicexamination similarly to case 1. Frozen samples, includingpericardial fluid and major organs, were also collected andstored at �80 8C.

2.2. PCR amplification

DNA was extracted from paraffinized intestine/stomach(cases 1 and 2) or fresh intestine (case 3) using acommercial kit (DNeasy, Qiagen, Valencia, CA) according

Fig. 1. Stomach from case 1. Small round (1–2 mm diameter) amphophilic

protozoa consistent with Cryptosporidium sp. are indicated by arrows.

Hematoxylin and Eosin staining. Bar = 20 mm.

Fig. 2. Small intestine from case 2. Along the small intestinal mucosa are

numerous protozoa consistent with Cryptosporidium sp (arrows). Some of

these protozoa have visible internal structures (arrowhead). A mixed

inflammatory cell population is within the lamina propria (*).

Hematoxylin and Eosin staining. Bar = 20 mm.

C. Griffin et al. / Veterinary Parasitology 170 (2010) 14–1916

to standard methods as described in the manual.Amplification using consensus primers for the Cryptos-poridium 18S rRNA gene was performed using previouslydescribed methods (Xiao et al., 1999). It should be notedthat a correction to this protocol is published separately,and is not listed as an erratum (Xiao et al., 2000). Directsequencing was performed. All products were sequencedin both directions.

2.3. Phylogenetic analysis

The sequences were compared to those in GenBank(National Center for Biotechnology Information, Bethesda,Maryland), EMBL (Cambridge, United Kingdom), and DataBank of Japan (Mishima, Shizuoka, Japan) databases usingBLASTN (Altschul et al., 1997).

Predicted homologous 753–813 nucleotide sequencesof representative cryptosporidial 18S rRNA availablefrom GenBank were aligned using three methods;ClustalW2 (Thompson et al., 1994), T-Coffee (Notredameet al., 2000), and MUSCLE (Edgar, 2004). Bayesiananalyses of each alignment were performed usingMrBayes 3.1 (Ronquist and Huelsenbeck, 2003). At the50 end, there were 4, 5, and 22 homologous nucleotidesmissing from the star tortoise (GenBank accession #AY120914), leopard gecko 1665 (AY120915), and Boa

2162 (AY268584) Cryptosporidium samples, and therewere also 14 nucleotides missing from the 30 end of theBoa 2162 Cryptosporidium sample. A general timereversible model with invariant + 4 gamma categoriesof rates was used. Four chains were run and statisticalconvergence was assessed by looking at the standarddeviation of split frequencies as well as potential scalereduction factors of parameters. The first 10% of1,000,000 iterations were discarded as a burn in.

Maximum likelihood (ML) analyses of each alignmentwere performed using PHYLIP (Phylogeny InferencePackage, Version 3.66) (Felsenstein, 1989), running eachalignment using the program dnaml with a general timereversible model, global rearrangements, five replicationsof random input order, and gamma plus invariant ratedistributions. The values for the gamma distribution weretaken from the Bayesian analysis. Theileria bicornis

(GenBank accession # AF499604), a non-coccidian api-complexan, and Goussia janae (AY043206), a non-Cryptos-

poridium coccidian, were used as the outgroup. To test thestrength of the tree topology, the alignment producing themost likely tree was then used for bootstrap analysis (200re-samplings) (Felsenstein, 1985).

3. Results

3.1. Pathology

3.1.1. Case 1

The gastric mucosa supported large numbers of smallround (1–2 mm diameter) amphophilic protozoa consis-tent with Cryptosporidium sp., with an eccentrically locateddense basophilic internal structure (Fig. 1). Minimalinflammation of lymphocytes and rare heterophils werein the lamina propria of the mucosa.

The lamina propria of the small intestinal mucosa andsubmucosa were diffusely infiltrated with lymphocytes,plasma cells, and heterophils with small numbers ofmacrophages. Within the lumen were mats of fungalhyphae multifocally admixed with inflammatory exudates.The fungal hyphae had internal septations, branching, andparallel cell walls. No further characterization of the fungalorganisms was done.

3.1.2. Case 2

No significant gross findings were noted. Tissues werecollected and processed for examination similarly totortoise 1. The small intestinal lamina propria wasinfiltrated with lymphocytes, plasma cells, and rarelyheterophils. Along the mucosa were numerous protozoaconsistent with Cryptosporidium sp., which were round,approximately 1–2 mm in diameter, and closely adhered tothe brush border (Fig. 2).

Fig. 3. Bayesian phylogenetic tree of T-Coffee alignment of homologous 753–813 base pairs of 18S rRNA nucleotide sequences. Theileria bicornis (GenBank

accession # AF499604), a non-coccidian apicomplexan, and Goussia janae (GenBank accession # AY043206), a non-Cryptosporidium coccidian, were used as

the outgroup. Confidence of the tree topology obtained is shown by Bayesian posterior probabilities to the left of the slash in bold and maximum likelihood

bootstrap values to the right. Areas of multifurcation are marked with arcs. Samples from this study are in bold. Site of infection, when known, is shown in a

column on the right. Cryptosporidium sp. sequences retrieved from GenBank include ‘‘Japanese grass snake kn732’’ (GenBank accession # AB222185), C.

varanii (AF112573), C. serpentis (AF093499), C. muris (AF093497), C. fragile (EU162751), ‘‘star tortoise 750’’ (AY120914), ‘‘leopard gecko 1665’’ (AY120915),

C. wrairi (AF115378), ‘‘Boa constrictor 2162’’ (AY268584), C. baileyi (AF093495), C. felis (AF112575), and C. parvum (AF164102).

C. Griffin et al. / Veterinary Parasitology 170 (2010) 14–19 17

3.1.3. Case 3

Grossly, the pericardial sac was thickened by edemaand contained 2.0 ml of cloudy, serous fluid. Thin strands offibrin were diffusely adhered to the epicardium andmultifocal white foci were observed on the surface ofthe ventricle. Cytological examination of the pericardialfluid revealed myriad spiral bacteria 6.0–10.0 mm in lengththat were distributed within a granular, proteinaceousbackground that were later found by direct sequencing of aconsensus 16S rRNA PCR product (Schuurman et al., 2004)to be a Helicobacter sp. (Stacy and Wellehan, 2010). In thesmall intestine, multiple sections had moderate, diffusehyperplasia of the mucosa. Various stages of numeroussmall (1.0–5.0 mm diameter) round protozoa consistentwith Cryptosporidium sp. were within the apical cytoplasmof mucosal epithelial cells and admixed with luminalmucus. Small numbers of heterophils multifocally infil-trated the submucosa.

3.2. PCR amplification

The product size was 770 nucleotides from tortoise 1when primer sequences were edited out, and 764nucleotide products from cases 2 and 3. The sequences

from cases 2 and 3 were identical. Sequences weresubmitted to GenBank under accession numbersGQ504268–GQ504270.

3.3. Phylogenetic analysis

BLASTN results for tortoise 1 showed the highest scorewith a Cryptosporidium from feces of an Indian star tortoise(Geochelone elegans) ‘‘750’’ (GenBank accession # Y09598)(Altschul et al., 1997). BLASTN results for tortoises 2 and 3showed equally highest scores with a Cryptosporidium

from a viper boa (Candoia asper) ‘‘938’’ (GenBank accession# AY120913) and another from a Japanese grass snake(Rhabdophis tigris) ‘‘kn732’’ (AB222185). These twosequences are identical. Although it was not found on aBLASTN search due to shorter sequence length, comparisonof the sequence from tortoises 2 and 3 with a shorter531 bp fragment from Cryptosporidium from feces of amarginated tortoise (Testudo marginata) ‘‘CrIT-20’’ (Gen-Bank accession # Y09598) found 100% homology over theavailable region.

A Bayesian tree using the T-Coffee alignment is shown(Fig. 3). The analysis identified that the Cryptosporidium

from the pancake tortoise and the second Russian tortoise

C. Griffin et al. / Veterinary Parasitology 170 (2010) 14–1918

formed a distinct clade, whereas the first Cryptosporidium

from the first Russian tortoise formed a more basal cladewith the Cryptosporidium from an Indian star tortoise(Geochelone elegans) ‘‘750’’. The intestinal Cryptosporidium

sp. segregate, forming a strongly supported monophyleticclade.

4. Discussion

In the minimal surveillance that has been done to date,there have been two distinct Cryptosporidium identified,and it is probable that additional species remain to bediscovered. These species are highly likely to differ in theirpathology. As an example, significant clinical differenceshave been found between C. serpentis and C. varanii. Whiletheir names may suggest specificity for snakes and lizards,respectively, this is not the case. C. serpentis has been foundin lizards (Xiao et al., 2004), and C. varanii has been foundin snakes (Plutzer and Karanis, 2007). This is not surprising,given the relationships of snakes and lizards. Evolutiona-rily, snakes branched off in the middle of the squamates, soif snakes are considered separate from other squamata,then lizards are not a monophyletic group (Vidal andHedges, 2005). The primary known clinical differencebetween C. serpentis and C. varanii is tropism for thestomach and intestine, respectively. It can be seen in thephylogenetic tree that C. serpentis clusters near C. muris,which also has gastric tropism.

One of the Cryptosporidium sp. in our study wasassociated with intestinal lesions in two different tortoisespecies, and one was associated with gastric tropism. Theintestinal tortoise Cryptosporidium sp. was found to becloser to C. varanii in the phylogenetic analysis, whereasthe gastric tortoise Cryptosporidium sp. was found to becloser to C. serpentis. Tissue tropism appears to be astronger indicator of relationships than host range. Thismay prove useful for prediction of site of infection ofunknown species lacking histologic data; Cryptosporidium

‘‘Boa constrictor 2162’’ would be expected to be tropic forintestine. Knowledge of site of infection of a givenCryptosporidium sp. is also important for diagnosticsampling. Gastric lavage was found to be a better samplefor detection of C. serpentis than cloacal swabs (Graczyket al., 1996); this would not be expected for a species tropicfor the intestine.

In general, gastric cryptosporidiosis in squamates(O’Donoghue, 1995; Oros et al., 1998) results in mucosalhyperplasia. In the Russian tortoise with gastric cryptos-poridiosis, neither hyperplasia nor atrophy was noted,with very minimal inflammation of the gastric sections.There is a variation in the lesions described with intestinalcryptosporidiosis in squamates. Small intestinal mucosalhyperplasia has been described (Kuroki et al., 2008; Terrellet al., 2003). Intestinal cryptosporidiosis was associatedwith severe inflammatory infiltrates and necrosis in roughgreen snakes (Opheodrys aestivus), whereas lesions in agarter snake had much milder inflammation present(Brower and Cranfield, 2001). Some of the variation inintestinal hypertrophy may be due to the concurrentanorexia commonly associated with the infection. Severalstudies have demonstrated fasting-induced atrophy of the

small intestine mucosa and rapid hypertrophy withrefeeding in reptiles (Lignot et al., 2005; Secor, 2005;Christel et al., 2007). Neither atrophy of the intestinalsection nor hyperplasia was identified in the Russiantortoise with intestinal cryptosporidiosis, while intestinalmucosal hyperplasia was present in the pancake tortoise.

The host ranges of tortoise Cryptosporidium species arenot known. It appears that the species that is tropic for thestomach can at least infect Russian tortoises, Hermann’stortoises, and Indian star tortoises, and the intestine-tropicspecies can infect Russian tortoises, pancake tortoises, andmarginated tortoises. Their zoonotic potential is notknown. Cryptosporidium serpentis has been shown not toinfect the only mammal that has been experimentallyinvestigated, mice (Mus musculus) (Fayer et al., 1995).Black rat snakes (Elaphe obsoleta) have been found not to besusceptible to several mammal Cryptosporidium sp.,including C. parvum, making carriage of zoonotic speciesunlikely (Graczyk and Cranfield, 1998a). However, experi-mental infection of snakes with Cryptosporidium oocystsfrom an Indian star tortoise was found to cause gastricdisease in snakes (Graczyk and Cranfield, 1998a). TheIndian star tortoise oocysts in that study were notidentified, and it is not known if it was one of the tortoisegenotypes found in the present study.

With the continued increase of tortoises in the pettrade, environmentally stable organisms that are capableof causing persistent infections pose a risk. Cryptospor-

idium sp. may play a role in the failure of some tortoises tothrive and grow. Cryptosporidium should also be consid-ered a possible etiologic agent in chronic gastrointestinaldisease of pancake and Russian tortoises, especially thosethat show subsequent weight loss, lethargy and mortality.

Conflict of interest

The authors have no conflicting interests.

Acknowledgement

An abstract was presented at the Conference of theAssociation of Reptilian and Amphibian Veterinarians, LosAngeles, October 2008.

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